In vivo genome editing using a high-efficiency TALEN system

Although zebrafish is an important animal model for basic vertebrate biology and human disease modelling, rapid targeted genome modification has not been possible in this species; here a technique based on improved artificial transcription activator-like effector nucleases (TALENs) allows precise sequence modifications at pre-determined genomic locations. Although well established as a model for studying basic vertebrate biology and human disease, the zebrafish has been lacking in one important respect: rapid targeted genome modification has not been possible in this species. Now, Stephen C. Ekker and colleagues report an approach that brings targeted genome editing and functional genomic applications to the zebrafish. The technique is based on improvements in artificial transcription activator-like effector nucleases (TALENs). Using this system, the authors precisely modify sequences at predefined genomic locations — for example, adding a custom-designed EcoRV site, which is successfully transmitted through the germline. The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease with a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications1,2,3,4,5. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show that this enhanced TALEN toolkit has a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino-based targeted gene knockdowns6. With this updated TALEN system, we successfully used single-stranded DNA oligonucleotides to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair, including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.